This invention relates to screening assays. Specifically, this invention relates to screening assays to identify compounds that effect signal transducing protein phosphorylation cascades by targeting the membrane anchor of long chain fatty acid modified trigger proteins. More specifically, this invention relates to drug screens to identify drugs that effect ligand mediated signal transducing protein phosphorylation cascades (for example; Ras/Rho mediated signal transducing protein phosphorylation cascades) by effecting the long chain fatty acid dependent membrane anchorage of long chain fatty acid modified trigger proteins (for example; prenylated or palmitoylated trigger proteins).
Multicellular organisms have a multiplicity of specialized cells functioning in cooperation. Communication in the organism to coordinate cooperative functions is mediated by a number of well known messengers, for example: hormones, neurotransmitters, cytokines, etc. The messengers communicate by binding a receptor at the cell membrane. The receptor binding activates a desired cell function. Alternatively, and/or additionally, cell functions are activated in response to signals generated and received intracellularly. The identification and characterization of the molecules involved, and the elucidation of mechanisms whereby such molecules cooperatively regulate the functions and link the signals to those functions, is one of the great scientific advances of the past few decades.
Membrane associated receptors are exquisitely specific for ligands (signal molecule) such as hormones. Any one cell exhibits a multiplicity of these receptors, the composition of which defines the types of signals to which that cell responds functionally. The receptors are often associated within the membrane superstructure with trigger proteins. When such a receptor binds the signal molecule it activates the trigger protein. These trigger proteins are in turn linked to initiate a cascade of regulatory molecules leading to the appropriate function of the cell that is activated by the hormone. A review of such a receptorxe2x86x92triggerxe2x86x92cascadexe2x86x92function phenomenon is provided in Denhardt, D. T., Signal-transducing Protein Phosphorylation Cascades Mediated by Ras/Rho Proteins in the Mammalian Cell: The Potential for Multiplex Signaling, 318 Biochem. J. 729-747 (1996) (xe2x80x9cDenhardtxe2x80x9d), the entirety of which is incorporated herein by reference.
Each trigger protein associates with specific membranes in the cell and the activity, and also often the specificity, of the protein is dependent on the trigger protein being associated with the correct membrane. To accomplish this the protein is often modified by one or more long chain fatty acids. Sometimes the protein is also carboxymethylated, glycosylated, cleaved, etc. but it appears that the most essential modification for directing many proteins to insert into the proper membrane is the long chain fatty acid modification(s).
One significant example set are cellular Ras proteins, extensively studied because mutated forms, oncogenic Ras proteins, are involved in the generation of many types of cancer. Ras is modified at its carboxy-terminus with a prenyl group and at a mid-chain Cys with a palmitoyl group. Conceptually, for Ras to be active it must be prenylated and anchored in the membrane. The prenylation of such trigger proteins as Ras, and many other related proteins, is reviewed in Omer, C A, and J. B. Gibbs, Protein Prenylation in Eukaryotic Microorganisms: Genetics, Biology, and Biochemistry, 11 Molecular Microbiology 219-225 (1994) (xe2x80x9cOmer and Gibbsxe2x80x9d), the entirety of which is incorporated herein by reference.
In brief, referring to FIG. 1, an immature trigger protein 1 is processed in the cell by the addition of a prenyl group 2 to a C-terminal cysteine. This prenylation reaction, catalyzed by a prenylprotein-transferase 3, is one of the processing reactions that results in the production of a mature prenylated trigger protein 4. Prenylated trigger protein 4 attaches to a trigger protein anchorage site 6 in a cellular membrane 5 resulting in a membrane anchored prenylated trigger protein 4xe2x80x2.
Membrane anchored prenylated trigger protein 4xe2x80x2 is stable and fully mature, yet remains inactive until an effector 7 activates it via a signal 8 acting on membrane receptor 6. Signal 8 can arise on either face of the membrane or can be both intracellular and extracellular. Such a signal can be activating or inactivating. If activating, as illustrated in this figure, membrane anchored prenylated trigger protein 4xe2x80x2 becomes an activated prenylated trigger protein 9. Activated prenylated trigger protein 9 has an enzymatic or binding activity that launches a regulatory cascade 10 of reactions. The reactions of regulatory cascade 10 link activated prenylated trigger protein 9 to a cellular function 11. Cellular function 11 consists of one or a set of cellular actions that define the specialized function of the activated cell. This cascade activation system allows a single trigger event to regulate multiple cell actions that must work in concert to accomplish a particular cell function, such as cellular growth or division.
There are numerous examples of signal transducing regulatory trigger pathways of the type generalized in FIG. 1. Denhardt defines various mammalian trigger proteins (including the gene products of Ras superfamily, and other G-proteins or GTP-binding proteins) and associated function cascades.
Briefly:
the Ras family, including H-ras, N-ras, Ka-ras, and Kb-ras, partition to specific membranes depending on the type of prenylation and, among other functions, regulate cell cycling and adhesion (mutations of ras, known as oncogenic ras, are involved in the formation of some types of tumors);
the Rap family, found in granules of Golgi and ER antagonize ras function (Rap 1A, also known as Krev-1, antagonizes the Kras oncogene);
the Ra1 family, including Ra1-a and Ra1-B appear to regulate the activity of exocytic and endocytic vesicles;
the Rho family, which includes Rho-A, Rho-B, Rho-C, Rac-1, Rac-2, CDC42, Rho-G, and TC1O, play dynamic roles in the regulation of the actin cytoskeleton and focal contacts mediating formation of filopodia and lamellipodia (Rac also controls NADPH oxidase activity in phagocytes);
the Ran proteins are involved in the transport of RNA and protein across the nuclear membrane;
the ARF/SAR proteins are important for vesicle formation and budding;
and the large and extensively studied
Rab/Ypt family are involved both in regulating intracellular vesicle trafficking between donor and acceptor membrane-enclosed compartments and in controlling the exocytosis and endocytosis of different types of vesicles.
In addition to the trigger proteinxe2x80x94cascade systems reviewed in Denhardt, other systems have also been described and extensively studied. For example, a family of myristoylated proteins are described that have a myristoyl group (C14:0) covalently attached, via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. The attachment is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) as a cotranslational modification. Compounds that block NMT activity have been shown to be potentially useful as anti-fungal, anti-viral, and anti-parasitic agents, and are therefore postulated to be useful for treating intracellular pathogens. A list of references and U.S. Patents detailing the myristoylation trigger protein system are found in table 1 each the entirety of which is incorporated herein by reference.
It appears that in order for most trigger proteins to become mature and capable of regulating a function cascade they must be long chain fatty acid modified and associated with the appropriate cell membrane anchor. Omer and Gibbs describe modification by prenylation of Ras superfamily encoded proteins, wherein, three enzymes are identified that mediate prenylation; farnesyl protein transferase, geranylgeranyl protein transferase type I, and geranylgeranyl protein transferase type II. These three enzymes, depicted generally in FIG. 1 as prenyl protein transferase 3, have become the target for the development of a number of drugs. Farnesyl protein transferase is the subject of at least 46 U.S. patents since 1992, listed herewith in appended Table 2, each the entirety of which is incorporated herein by reference.
In addition to prenylation, palmitoylation is essential to membrane anchorage of some of the RAS superfamily members listed in table 2, as well as many other membrane associated trigger proteins. Articles describing the palmitoylation of various other membrane associated cellular proteins and the relevance of palmitoylation to their cellular function, are listed in appended Table 3, each the entirety of which is incorporated herein by reference.
Inhibitors of farnesyl protein transferase block the maturation of farnesylated trigger proteins and prevent them from associating with the appropriate membrane. For example, drug developers look for compounds that block the activity of Oncogenic Ras (xe2x80x9cOncRasxe2x80x9d) activity without effecting normal Ras. To accomplish this they screen for compounds that preferentially block the farnesylation of OncRas and not normal Ras by farnesyl protein transferase. Since farnesylation is necessary for maturation and association of Ras or OncRas with the plasma membrane, a drug that blocks the farnesylation of OncRas necessarily blocks its activity.
Farnesyl protein transferase inhibitors are attractive to drug developers because of their potential for treatment on many types of cancer. OncRas, a mutated form of Ras, is directly implicated as the cause of most pancreatic tumors and more than 50% of colon cancer cases. Additionally, permanent activation of Ras by over-production of hormones and growth factors is implicated as the causative agent in tissue specific growth factor induced tumors. Besides tumor treatment, farnesyl protein transferase inhibitors as well as geranyl derivatized inhibitors are being developed for cholesterol reduction and treatment of atheroscleroses.
The main conceptual drawback of developing inhibitors of specific prenyl protein transferases is that these enzymes play significant critical roles in a multiplicity of normal cellular processes. Such a drawback may explain the difficulties encountered by drug developers who have developed inhibitors of these enzymes. Such difficulties include incomplete enzyme inhibition, toxicity, and non-specificity. In this regard, this application describes a method of identifying drugs that displace mature prenylated trigger protein from their membrane anchor site rather than inhibiting their prenylation by protein prenyl transferases.
It is first object of this invention to overcome the drawbacks and inconveniences of the prior art. In that regard, it is an object of the present invention to provide a drug screen, including the steps of: providing an assay material, the assay material including a specific membrane and a specific membrane anchored target protein, and the assay material having a known quantity of the specific membrane anchored target protein associated with the specific membrane.
Exposing the assay material to a compound that is being tested for the compound""s ability to disrupt membrane association of the specific membrane anchored target protein. Separating the assay material into a membrane fraction of the specific membrane and a cytosolic fraction of a balance of the assay material remaining after the specific membrane is removed. And, at least one of determining a fraction of the known quantity that is a released quantity of target protein in the cytosolic fraction and determining a fraction of the known quantity that is a non-released quantity of target protein in the membrane fraction.
It is another object of the present invention to provide a method of testing a compound for its effect on specific lipoprotein membrane anchorage, including the steps of: labeling the lipoprotein with a tag, exposing the membrane anchorage to the compound, and at least one of detecting a membrane-disassociated tag concentration and detecting a membrane associated tag concentration.
It is another object of the present invention to provide a drug screen, including the steps of: providing an assay material, the assay material including a specific membrane and at least one target anchored in the specific membrane, and the assay material having a determinable quantity of the target, exposing the assay material to a compound that is being tested for the compound""s ability to release membrane association of the target from the specific membrane, and at least one of determining a fraction of the determinable quantity that is a released quantity of the target and determining a fraction of the determinable quantity that is a non-released quantity of the target.
It is a feature that the drug screen further includes the step of labeling the specific membrane anchored protein with a detectable tag whereby the known quantity is trackable following fractionation by detecting the tag. Also, the step of determining a fraction of the known quantity that is a released quantity of target protein in the cytosolic fraction is accomplished by determining a quantity of the tag in the cytosolic fraction, and the step of determining a fraction of the known quantity that is a non-released quantity of target protein in the membrane fraction is accomplished by determining a quantity of the tag in the membrane fraction.
It is a further feature of the drug screen that the step of labeling is one selected from the group consisting of genetic labeling, metabolic labeling, chemical labeling, immunologic labeling, and labeling by identification of an intrinsic characteristic of the specific membrane anchored target protein, and the tag is at least one selected from the group consisting of green fluorescent protein, Alkaline Phosphatase, Horseradish Peroxidase, Urease, xcex2-galactosidase, CAT, Luciferase, an immunogenic tag peptide sequence, an extrinsically activatable enzyme, an extrinsically activatable toxin, an extrinsically activatable fluor, an extrinnsicly activatable quenching agent, a radioactive element, and an antibody.
It is a further feature of the drug screen that the specific membrane is at least one of a plasma membrane, a nuclear membrane, a endoplasmic reticulum, a golgi, and a vesicle.
It is a further feature of the drug screen that the target protein has at least one long chain fatty acid group.
It is a further feature of the drug screen that the long chain fatty acid group is at least one of a prenyl, a myristoyl, and a palmitoyl.
It is a further feature of the drug screen that the prenyl is selected from the group consisting of a farnesyl, a geranyl, and a geranylgeranyl.
It is a further feature of the drug screen that the target protein is at least one selected from the group consisting of; the Ras family proteins, including H-ras, N-ras, Ka-ras, and Kb-ras, the Rap family proteins, the Ral family proteins, including Ra1-a and Ra1-B, the Rho family proteins, which includes Rho-A, Rho-B, Rho-C, Rac-1, Rac-2, CDC42, Rho-G, and TCIO, the Ran proteins, the ARF/SAR proteins, the Rab/Ypt family proteins, the family of myristoylated proteins; and the family of trimeric G-proteins.
It is another feature that the drug screen further includes the step of labeling the target with a detectable tag whereby the known quantity of target protein is trackable following fractionation by detecting the tag.
It is another feature that the drug screen further includes the step of providing a label gene in a cell whereby a release of the at least one target from the specific membrane causes the label gene to one of ceasing expression and commencing expression.
In summary, the present invention is a drug screening assay for identifying compounds for their potential effects on the long chain fatty acid dependent intracellular membrane anchorage sites of lipoproteins. Membranes-anchoring target are incubated in the presence of the compound to be tested and the proportion of the target that is released by the compound is detected and quantitated.
The benchmark for this assay are the prenylated proteins such as the farnesylated oncogenic ras trigger proteins which are displaced from their anchorage at the intracellular plasma membrane by Farnesyl Thiosalicylic Acid (FTS) and derivatives of FTS. The assay is adaptable to flexibly assay a large variety of anchored targets using a wide range of labeling and detection techniques in test wells, tissue culture, and in animals, as injected cells or transgenics, thereby directly addressing a wide range of pharmacologically relevant questions.
The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings, in which like reference numerals designate the same elements.